CARS is a 3rd order non-linear optical interaction that has been used for chemical analysis, combustion and flow diagnostics, and is now being applied (by many different groups worldwide) to biological microscopy. CARS signals are always generated in concert with non-resonant background (NRB).
Fluorescence microscopy has allowed a great deal of cell structure and function to be studied, but it has some inherent drawbacks. It relies on tagging proteins with fluorescent dyes or genetically encoded fluorescent proteins, which can alter the targeted protein functions. Furthermore, fluorescent dyes bleach under illumination, which limits sensitivity and creates reactive species in the cell. CARS avoids these problems: it does not cause bleaching, as it is a parametric process which leaves no energy in the target molecule; and the signal depends on vibrational resonances, so it allows chemical species to be identified without tagging. There has therefore been an intense research effort over the last decade exploring the application of CARS to microscopy.
However, CARS has some drawbacks of its own. It is more complex to implement than fluorescence, as three beams (called the pump, probe and Stokes beams) of at least two different frequencies must be combined in the sample to generate the fourth, anti-Stokes, beam. Also, all media have a third order non-linear optical response, so the resonant signal is always generated along with a non-resonant background signal (NRB). As CARS is a coherent process the two signals are not simply additive; they will add and subtract depending on their relative phase. This distorts line shapes and makes spectral analysis difficult. Also, CARS requires pulsed lasers with high peak energies, and under these conditions the NRB becomes large, obscuring weak resonant signals. As a consequence, much of the research into CARS microscopy has been directed at developing ways to remove the NRB.
Most of the CARS microscopy studies to date have looked at lipid rich structures, as the high density of C-H vibrations gives strong signals that can be distinguished against the NRB. However, the line distortion and decreased signal-to-background due to NRB has so far prevented full realisation of the initial promise of CARS microscopy: that is, chemical specificity without tagging (as in spontaneous Raman scattering).
Despite CARS being a very useful technique in biology, the vast majority of CARS microscopes in use today are hand-built systems: complexity, expense and the specialist expertise required to operate them has (until recently) hindered development of a commercial product. Existing NRB removal schemes require expensive laser systems with near transform limited pulses, along with complex instruments such as spatial light modulators (SLM's).